Field of the Invention
This invention relates to compositions and methods for delivery of biomolecules into cells.
Description of the Related Art
Recently, as the fields of gene therapy and molecular biology have developed rapidly, an urgent need has emerged to effectively deliver biomolecules, such as proteins, nucleic acids, protein analogs, nucleic acid analogs, including oligonucleotides such RNA, DNA and analogs thereof, peptides, polypeptides, proteins, antibodies, hormones, small molecules, antiviral agents and the like into cells or tissues. Many therapeutic, research, and diagnostic applications rely upon the efficient transfer of biologically active molecules into cells, tissues, and organs.
Particularly problematic in the delivery of biomolecules is the delivery of negatively charged molecules, such as polyribonucleic acids, and polydeoxyribonucleic acids and analogs thereof, due to their size and charge. To be useful in therapeutic or research, however, an effective amount of the biomolecule, e.g., polynucleotides, oligonucleotides or the like, must be delivered into the target cells or tissue. At the same time, when used in a therapeutic setting, nucleic acid delivery methods should minimize immune responses or cytotoxicity to the host Finally, availability of large-scale production of delivered materials, or vehicles, is desirable.
Current methods for delivery of negatively charged biomolecules include viral-based delivery systems and non-viral based delivery systems.
Virus-mediated delivery of nucleic acids is known in the art. Viral based gene nucleic acid delivery systems utilize retrovirus, adenovirus, and adeno-associated viruses. Virus-mediated nucleic acid delivery has drawbacks, however, including narrow range of cell infectivity, the elicitation of immune responses, and difficulty of large-scale production of viral vectors. (Yibin Wang et al., DDT. 5(1), 2000; Joanne T. Douglas. et al., Science & medicine 44-52 (March/April), 1997).
Non-viral delivery systems include systems such as liposomes, polymers, calcium phosphate, electroporation, and micro-injection techniques (Saghir Akhtar et al., Adv. Drug Deliv. Rev. 44:3-21; Irina Lebedeva et al., Eur. J. Pharm. Biopharm. 50:101-119, 2000; Ch. Garcia-Chaumont et al., Pharmacol. Ther. 76:151-161, 2000). Ease of preparation and large-scale production have made the use of non-viral vectors a popular option for gene therapy. (Colin W. Pouton et al., Adv. Drug Deliv. Rev. 46:187-20, 2001).
Among the non-viral vectors developed to date, liposomes are the most frequently used gene transfer vehicle and are available commercially. Many liposomes are cationic. Cationic liposomes, complexed with nucleic acids or analogs thereof, electrostatically interact with the cell surface, and the complexes are then endocytosed into the cell cytoplasm. The cationic nature of the liposomes facilitates passage of negatively charged biomolecules such as polynucleotides across the cell wall. However, while cationic liposomes mediate gene delivery effectively into cells in vitro, gene delivery in an in vivo system is quite limited as compared to viral vectors. Furthermore, the efficiency of gene delivery using cationic liposomes is generally dependent on the size of nucleic acids, and the cell line, even in an in vitro system. The major drawback of cationic liposomes, however, is their known cytotoxicity to cells (Saghir Akhtar et al., Adv. Drug Deliv. Rev. 44:3-21, 2000: Irina Lebedeva et al., Eur. J. Pharm. Biopharm. 50:101-119, 2000).
Other cationic systems, such as cationic polymers, have been used to increase the efficiency of biomolecule delivery into cells. Polymers with numerous, positively-charged amine groups are able to bind strongly with nucleic acids, and also interact with the cell, so that the required amount of the polymers as compared to that of cationic liposomes can be reduced. However, cytotoxicity and insolubility of cationic polymers in aqueous solutions are drawbacks that limit the usefulness of cationic polymers as an effective gene delivery vehicle (Dan Luo et al., Nat. biotech. 18:33-37; Saghir Akhtar et al., Adv. Drug Deliv. Rev. 44:3-21, 2000).
Another non-viral system for the delivery of biomolecules relates to the addition of a covalently linked antibody to the oligonucleotide. The antibody mediated approach to delivery of biomolecules falters due to the therapeutic being shuttled down the endosomal pathway leading to ultimate degradation of the biomolecule.
Accordingly, there exists a need for improved biomolecule delivery systems.